Evolutionary perspectives on multiresistance beta-lactamase transposons.

A series of intragenic DNA probes, encoding the major part of the transposase resolvase and inverted repeats of transposons Tn3, Tn21, and Tn2501, were used in hybridization assays for homologous DNA sequences in 18 transposons studied. The tnpA and tnpR probes detected extensive homology with Tn3-like and Tn21-like elements for 11 transposons. This high degree of homology was confirmed with the 38- and 48-base-pair inverted-repeat oligonucleotide probes of Tn3, Tn21, and Tn2501. The Southern-type gel hybridization experiments localized the tnpA-homologous sequences on the physical DNA maps constructed. The genetic and physical maps of the transposons were compared, as were their nucleic acid sequence homologies. These comparisons suggested a subfamily of mobile elements distinct from but related to the Tn21 group. Based on these results, an evolutionary model is proposed and a pedigree is presented for the genesis of multiresistance beta-lactamase transposons.     

Structural and evolutionary relationships of beta-lactamase transposons from Staphylococcus aureus

A comparison of the beta-lactamase elements detected on three classes of large plasmids together with the chromosomes of penicillin-resistant Staphylococcus aureus revealed substantial physical and genetic relatedness. In most cases, beta-lactamase production could be associated with the presence of a DNA segment of approximately 6.7 kb. Analysis showed that the plasmid-borne determinants constitute nearly identical transposons or transposon-like elements. An element indistinguishable from one of these, Tn4002, which is carried by the pSK1 family of plasmids in clinical isolates from Australian hospitals, was also identified on the staphylococcal chromosome and is implicated in an evolutionary cycle of transposition between chromosomal and extrachromosomal sites in Australian strains of multiresistant S. aureus.     

Oligonucleotide probes for the detection of TEM-1 and TEM-2 beta-lactamase genes and their transposons

We describe the use of molecular probes to detect the TEM-type beta-lactamase genes. As a general probe, we prepared a 656 base pair restriction fragment, entirely within the TEM structural gene. This probe was specific for the TEM family, hybridizing only with TEM-1 and TEM-2. The TEM-1 and TEM-2 beta-lactamases differ by only one amino acid. We synthesized two oligonucleotides whose central bases correspond to this difference. The use of these oligonucleotides enables us to discriminate between TEM-1 and TEM-2 genes. Using oligonucleotides homologous to parts of Tn3, we also monitored the presence of TnA-like transposons in bacteria harboring different beta-lactamase genes. Only the TEM-1 and TEM-2 genes were found to be on transposons with terminal sequences identical to those of Tn3. All hybridization experiments were performed with both dot-blot and colony-hybridization techniques, and the suitability of these two methods for epidemiological studies is compared.     

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.     

Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes

Summary Molecular cloning of DNA fragments between 1.5 and 8kb from BamHI, EcoRI, HindIII, SalI, or Sau3A digests permitted the isolation of structural genes coding for TEM-1, ROB-1, OXA-1, OXA-3, OXA-4, OXA-5, PSE-1, PSE-2, PSE-3, PSE-4, CARB-3, CARB-4, AER-1, and LCR-1 -lactamases. Ampicillin-resistant clones were selected and it was confirmed that they contained the respective -lactamase genes by isoelectric focusing. Detailed physical maps of 14 different recombinant plasmids were constructed using 8 restriction endonucleases. Plasmid deletions and lacZ fusions were used to localize the -lactamase structural genes. DNA probes were constructed for the TEM01, ROB-1, OXA-1, and PSE-1 genes. Under conditions of high stringency, hybridization was observed between the genes for TEM-1 and TEM-2 or TLE-1, OXA-1 and OXA-4, and PSE-1 and PSE-4 or CARB-3, while the ROB-1 gene probe showed no cross-hybridization. Such bla gene probes should facilitate studies of -lactamase molecular epidemiology.     

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.     

Crystal structure and kinetic analysis of beta-lactamase inhibitor protein-II in complex with TEM-1 beta-lactamase

The structure of the 28 kDa beta-lactamase inhibitor protein-II (BLIP-II) in complex with the TEM-1 beta-lactamase has been determined to 2.3 A resolution. BLIP-II is a secreted protein produced by the soil bacterium Streptomyces exfoliatus SMF19 and is able to bind and inhibit TEM-1 with subnanomolar affinity. BLIP-II is a seven-bladed beta-propeller with a unique blade motif consisting of only three antiparallel beta-strands. The overall fold is highly similar to the core structure of the human regulator of chromosome condensation (RCC1). Although BLIP-II does not share the same fold with BLIP, the first beta-lactamase inhibitor protein for which structural data was available, a comparison of the two complexes reveals a number of similarities and provides further insights into key components of the TEM-1-BLIP and TEM-1-BLIP-II interfaces. Our preliminary results from gene knock-out studies and scanning electron microscopy also reveal a critical role of BLIP-II in sporulation.     

Combinatorial genetic evolution of multiresistance

The explosion in genetic information, whilst extending our knowledge, might not necessary increase our conceptual understanding on the complexities of bacterial genetics, or why some antibiotic resistant genotypes such as blaCTX-M-15 and blaVIM-2 appear to dominate. However, the information we have thus far suggests that clinical isolates have 'hijacked' plasmids, primarily built of backbone-DNA originating from environmental bacteria. Additionally, the combinatorial presence of other elements such as transposons, integrons, insertion sequence (IS) elements and the 'new' ISCR (IS common region) elements have also contributed to the increase in antibiotic resistance - an antibiotic resistant cluster composing four or five genes has become commonplace. In some instances, the presence of antibiotics themselves, such as fluoroquinolones, can mediate a bacterial SOS cell response, subsequently amplifying and/or augmenting the transfer of large genetic entities therefore, potentially promoting long-term detrimental effects.     

Crystal structure of a cold-adapted class C beta-lactamase

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.     

Replication of staphylococcal multiresistance plasmids

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.     

Conservation in structure of TOC1 transposons from Chlamydomonas reinhardtii.

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.     

Coral and sea urchin assemblage structure and interrelationships in Kenyan reef lagoons

Patterns of hard coral and sea urchin assemblage structure (species richness, diversity, and abundance) were studied in Kenyan coral reef lagoons which experienced different types of human resource use. Two protected reefs (Malindi and Watamu Marine National Parks) were protected from fishing and coral collection, but exposed to heavy tourist use. One reef (Mombasa MNP) received protection from fishermen for one year and was exploited for fish and corals prior to protection and was defined as a transitional reef. Three reefs (Vipingo, Kanamai, and Diani) were unprotected and experienced heavy fishing and some coral collection. Protected and unprotected reefs were distinct in terms of their assemblage structure with the transitional reef grouping with unprotected reefs based on relative and absolute abundance of coral genera. Protected reefs had slightly higher (p<0.01) coral cover (23.6 ± 8.3 % ± S.D.) than unprotected reefs (16.7 ± 8.5), but the transitional reef had the highest coral cover (30.8 ± 6.4) which increased by 250% since measured in 1987: largely attributable to a large increase inPorites nigrescens cover. Protected reefs had higher coral species richness and diversity and a greater relative abundance ofAcropora, Montipora andGalaxea than unprotected reefs. The transitional reef had high species richness, but lower diversity due to the high dominance ofPorites. Sea urchins showed the opposite pattern with highest diversity in most unprotected reefs. Coral cover, species richness, and diversity were negatively associated with sea urchin abundance, but the relative abundance ofPorites increased with sea urchin abundance to the point wherePorites composed >90% of the coral cover at sites with the highest sea urchin abundance. Effects of coral overcollection was only likely for the genusAcropora (staghorn corals). A combination of direct and indirect effects of human resource use may reduce diversity, species richness, and abundance of corals while increasing the absolute abundance of sea urchins and the relative cover ofPorites.     

Probing beta-lactamase structure and function using random replacement mutagenesis.

A new analytical mutagenesis technique is described that involves randomizing the DNA sequence of a short stretch of a gene (3-6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein. Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical. We applied this method to 66 codons of the gene encoding TEM-1 beta-lactamase in 19 separate experiments. We found that TEM-1 beta-lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild-type enzyme. We also found a few exceptional regions where only a few random sequences function. Examination of the X-ray structures of homologous beta-lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein. DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related beta-lactamases.     

Crystal structure of the class D beta-lactamase OXA-10

We report the crystal structure of a class D beta-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 A resolution. There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C beta-lactamases. Furthermore, the structure suggests the unique, cation mediated formation of a homodimer. Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme. Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively). Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for beta-lactam hydrolysis. Clinical variants of OXA-10 are also discussed in light of the structure.     

Role of primary structure and disulfide bond formation in beta-lactamase secretion

Plasmid pBR322-encoded beta-lactamase was shown to contain a single disulfide bond, which caused the protein to migrate faster in sodium dodecyl sulfate-polyacrylamide gels than the fully reduced form. A similar difference in mobility of the in vitro synthesized precursor before and after reduction indicates that it also contained a disulfide bond. Formation of the disulfide bond in vivo, however, occurred concomitant with processing. In vivo accumulation of the precursor by inhibition of secretion did not allow disulfide bond formation to occur. This result is consistent with post-translational translocation of the precursor. Synthesis of a fragment of beta-lactamase lacking the carboxy terminus was obtained by insertion of a foreign DNA segment into the PstI site of bla. Processing and secretion of the protein did not appear to be greatly affected, indicating that the carboxy terminus is not required for secretion.     

Toluene transposons Tn4651 and Tn4653 are class II transposons

The toluene degradative transposon Tn4651 is included within another transposon, Tn4653, and both of these elements are members of the Tn3 family. The tnpA gene product of each element mediates formation of cointegrates as intermediate products of transposition, and the tnpS and tnpT gene products encoded by Tn4651 take part in resolution of both Tn4651- and Tn4653-mediated cointegrates. Sequence analysis demonstrated that Tn4651 and Tn4653 have 46- and 38-base-pair terminal inverted repeats, respectively, and that both elements generate 5-base-pair duplication of the target sequence upon transposition. Complementation tests of the Tn4651- and Tn4653-encoded transposition functions with those of Tn3, Tn21, and Tn1721 showed that (i) the trans-acting transposition functions encoded by Tn4651 were not interchangeable with those encoded by the four other transposons, (ii) the Tn4653 tnpA function was interchangeable with the Tn1721 function, and (iii) Tn4653 coded for a resolvase (tnpR gene product) that complemented the tnpR mutations of Tn21 and Tn1721. The Tn4653 tnpR gene was located just 5' upstream of the tnpA gene and shared extensive sequence homology with the Tn1721 tnpR gene. The res region was located adjacent to the tnpR gene, and sequence analysis indicated that failure of the Tn4653 tnpR product to resolve the Tn4653-mediated cointegrates is ascribed to an incomplete structure of the res region.